Although the Lyme disease spirochete, Borrelia burgdorferi, is difficult to recover from infected human tissues and fluids, we believe it is likely that persistent live spirochetes or spirochetal fragments are driving the illness throughout much of its course. Evidence for this view includes the responsiveness of many patients to antibiotics even after long-term infection, the occasional sightings of spirochetes in affected tissues, the antigen-specific proliferation of lymphocytes from these patients, and time-dependent variations in the antibody responses to spirochetal antigens m some individuals. However, the recovery of spirochetes from infected tissues by culture is only occasionally possible, and in direct methods based on a strong, specific immunological response have so far demonstrated a fundamental lack of sensitivity and specificity. We propose to use the polymerase chain reaction (PCR) as a diagnostic tool for the detection of the Lyme disease spirochete, and to monitor Lyme disease patients before, during, and after antibiotic therapy. Our expectation is that PCR will serve as a valuable adjunct to existing methods for the diagnosis and monitoring of Lyme disease. However, the greatest asset of PCR, high sensitivity, is tempered by a dangerous predilection for producing false positives, made worse because the products of the reaction itself (amplicons) may serve as carried-over substrates for future reactions. In addition to employing recommended procedures for reducing amplicon carryover, we will address this problem by: 1) having results verified in two outside laboratories, 2) employing amplicon sterilization techniques on a regular basis, and 3) using multiple genetic target loci for detection of the organism. Aside from addressing false positives, we feel the latter """"""""multi-locus"""""""" approach will be extremely useful for highlighting genetic differences between spirochetal isolates from different geographic areas, and even more importantly, for avoiding false negative results due to such differences when these methods are used on infected tissues. Thus, having set the stage with sterilization techniques and multiple genetic targets, and armed with preliminary data from old ticks, mice, and human clinical specimens, we are equipped to proceed with trials of this diagnostic technology for the detection and monitoring of B. burgdorferi infections in humans.

National Institute of Health (NIH)
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Research Project (R01)
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Arthritis and Musculoskeletal and Skin Diseases Special Grants Review Committee (AMS)
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Mayo Clinic, Rochester
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